BMP-3 antibodies and related methods

ABSTRACT

The present invention provides compositions and methods for promoting bone growth. The present invention also provides methods of screening compounds that modulate BMP-3A or BMP-3B activity. The compositions and methods provided herein are useful in modulating bone growth.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/092,353, filed onMar. 28, 2005, which claims the benefit of U.S. Provisional ApplicationNo. 60/557,100, filed Mar. 26, 2004, the specifications of which areincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Bone morphogenetic proteins (BMPs) are members of the transforminggrowth factor-β superfamily. Many BMPs are produced in bone and modulateosteogenic activity, which suggests that these proteins are involved inbuilding bone mass. Members of the BMP family include BMP-2 and BMP-3.BMP-2 has been implicated in multiple functions associated with boneformation and growth. For example, the protein induces differentiationof osteoprogenitor cells into osteoblasts, and to enhance healing ofbone fractures.

BMP-3 is also termed BMP-3A. A closely related protein termed BMP-3B isalso known as growth and differentiation factor 10 (GDF-10). These twoproteins show great amino acid sequence similarity in the regioncorresponding to the mature (fully processed) form, corresponding theC-terminal portion of the unprocessed protein. The differences betweenthe sequences of BMP-3A and BMP-3B are located mainly outside the maturepeptide regions, with the propeptides showing significant sequencedivergence. BMP-3A was originally purified from bone as osteogenin andis particularly abundant in demineralized bone. Recombinant BMP-3Ashowed no osteogenic activity and instead was reported to antagonize theosteogenic activity of BMP-2. Zhu et al., Chin J. Biotechnol. 1999;15(3): 153-8. This result was supported by an observed increase in bonedensity and volume in a BMP-3A −/− knock-out mouse. Daluiski et al.,Nat. Genet. 2001 January; 27(1):84-8.

In view of the above findings, a need exists for a manner of regulatingBMP-3A activity, particularly in individuals who are in need ofincreased bone mass or increased bone growth, such as individuals withosteoporosis or bone fractures.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides BMP-3 propeptides.As disclosed herein, BMP-3 propeptides bind to mature BMP-3 andcompetitively inhibit binding to Type II receptor. Accordingly, BMP-3propeptides may be used to inhibit BMP-3-Type II receptor mediatedsignaling. BMP-3 propeptides may be used for treating bone disorders,and particularly those disorders where increases in bone density orvolume are desirable, as in the treatment of osteoporosis and bonefractures. Additionally, such propeptides may inhibit other members ofthe BMP family and will be useful in the treatment of additionaldisorders. Likewise, the mature BMP-3A and 3B polypeptides mayparticipate in other biological processes, and a BMP-3 propeptidedisclosed herein may be used to antagonize BMP-3A and 3B in any BMP-3related process. Examples of BMP-3 propeptides include the propeptidesof BMP-3A and BMP-3B, as well as any functional variants thereof. Incertain cases, the BMP-3 propeptide inhibits the interaction between amature BMP-3 polypeptide and an ActRIIa polypeptide. In other cases, theBMP-3 propeptide inhibits any signaling mediated by interaction betweena mature BMP-3 polypeptide and an ActRIIa polypeptide. Optionally, aBMP-3 propeptide derepresses or increases bone growth. Additionally, thedisclosure provides antibodies that bind a mature BMP-3 peptide in amanner similar to a BMP-3 propeptide. Such antibodies may also be usedto treat bone disorders or other BMP-3 related disorders.

In certain aspects, the disclosure provides pharmaceutical preparationsfor promoting bone growth and/or inhibiting bone loss. Such preparationsmay comprise a BMP-3 propeptide that binds to a mature BMP-3 polypeptideand a pharmaceutically acceptable carrier. In certain cases, the BMP-3propeptide inhibits the interaction between a mature BMP-3 polypeptideand an ActRIIa polypeptide. In other cases, the BMP-3 propeptideinhibits any signaling mediated by interaction between a mature BMP-3polypeptide and an ActRIIa polypeptide. Optionally, a BMP-3 propeptidederepresses or increases bone growth. Preferably the BMP-3 propeptidebinds to a mature BMP-3 with a KD less than 1 micromolar or less than100, 10 or 1 nanomolar. A BMP-3 propeptide for use in such a preparationmay be any of those disclosed herein, such as a polypeptide having anamino acid sequence of SEQ ID NO:1 or 2 or having an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an aminoacid sequence of SEQ ID NO:1 or 2. A BMP-3 propeptide may include afunctional fragment of a natural BMP-3 propeptide, such as onecomprising at least 10, 20 or 30 amino acids of SEQ ID NO:1 or 2. ABMP-3 propeptide will generally not contain a full-length or functionalportion of a mature BMP-3 polypeptide, and preferably a BMP-3 propeptidewill include no more than 50, 40, 30, 20, 10 or 5 amino acids of amature portion of a BMP-3 polypeptide. A BMP-3 propeptide may include analteration in the amino acid sequence relative to a naturally occurringBMP-3 propeptide. The alteration in the amino acid sequence may alterglycosylation of the polypeptide when produced in a mammalian, insect orother eukaryotic cell or alter proteolytic cleavage of the polypeptiderelative to the naturally occurring BMP-3 polypeptide. A BMP-3propeptide may be a fusion protein that has, as one domain, a BMP-3propeptide (including any of the various truncations or variationsdescribed herein) and one or more additional domains that provide adesirable property, such as improved pharmacokinetics, easierpurification, targeting to particular tissues, etc. For example, adomain of a fusion protein may enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. A BMP-3 propeptide fusion proteinmay include an immunoglobulin Fc domain or a serum albumin. A fusionprotein may include a purification subsequence, such as an epitope tag,a FLAG tag, a polyhistidine sequence, and a GST fusion. Optionally, aBMP-3 propeptide includes one or more modified amino acid residuesselected from: a glycosylated amino acid, a PEGylated amino acid, afarnesylated amino acid, an acetylated amino acid, a biotinylated aminoacid, an amino acid conjugated to a lipid moiety, and an amino acidconjugated to an organic derivatizing agent. A pharmaceuticalpreparation may also include one or more additional compounds such as: acompound that inhibits bone resorption, a compound that stimulates boneformation, and/or a compound that increases bone mineral density. Forexample, the preparation may include a bisphosphonate. Preferably, apharmaceutical preparation is substantially pyrogen free. Preferably, apharmaceutical composition comprising a BMP-3 propeptide will notinclude, as a separate component, an active mature BMP-3 protein.

In certain aspects, the disclosure provides packaged pharmaceuticalscomprising a pharmaceutical preparation described herein and labeled foruse in promoting increased bone density or growth or diminishing orpreventing bone loss or demineralization in a human or non-humanpatient.

In certain aspects, the disclosure provides nucleic acids encoding aBMP-3 propeptide that do not encode a complete, translatable matureportion of a BMP-3. An isolated polynucleotide may comprise a codingsequence for a BMP-3 propeptide, such as described above. An isolatednucleic acid may include a sequence coding for a BMP-3 propeptide and asequence that would code for part or all of a mature portion, but for astop codon positioned within the mature portion or positioned betweenthe propeptide and the mature portion. For example, an isolatedpolynucleotide may comprise a polynucleotide sequence coding for apolypeptide selected from the group consisting of SEQ ID NO:7 and 8,said isolated polynucleotide further comprising a transcriptiontermination codon at least three hundred nucleotides before the3′-terminus. Nucleic acids disclosed herein may be operably linked to apromoter for expression, and the disclosure provides cells transformedwith such recombinant polynucleotides. Preferably the cell is amammalian cell such as a CHO cell.

In certain aspects, the disclosure provides methods for making a BMP-3propeptide. Such a method may include expressing any of the propeptideencoding nucleic acids disclosed herein in a suitable cell, such as aChinese hamster ovary (CHO) cell. Such a method may comprise: a)culturing a cell under conditions suitable for expression of thepropeptide, wherein said cell is transformed with a BMP-3 propeptideexpression construct; and b) recovering the propeptide so expressed.

In certain aspects, the disclosure provides methods for treating asubject having a disorder associated with insufficient bone mineraldensity, bone loss, bone damage or insufficient bone growth, the methodcomprising administering to the subject an effective amount of a BMP-3propeptide. The BMP-3 propeptide may be any of those disclosed herein.The subject may suffer from, for example, lower than normal bone mineraldensity, osteoporosis or a fracture. A method may includeco-administration with an additional agent, such as a bisphosphonate. Incertain embodiments, the disclosure provides methods for increasing bonedensity and/or bone volume in a subject, the method comprisingadministering a BMP-3 propeptide disclosed herein.

In further aspects, the disclosure provides methods for identifying anagent that increases bone density and/or bone volume. A method maycomprise: a) identifying a test agent that binds a mature BMP-3polypeptide competitively with a BMP-3 propeptide; and b) evaluating theeffect of the agent on bone growth. A test agent may be, for example, avariant BMP-3 propeptide, an antibody, or a small molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a BMP-3A propeptide amino acid sequence (SEQ ID NO: 1).

FIG. 2 shows a BMP-3B/GDF-10 propeptide amino acid sequence (SEQ ID NO:2).

FIG. 3 shows a BMP-3A precursor amino acid sequence (SEQ ID NO: 3). Thesignal peptide (residues 1-22) is underlined; the prodomain (residues23-362) is in bold, also referred to as SEQ ID NO: 1; and the matureprotein (residues 363-472) is shaded. The potential N-linkedglycosylation sites are boxed.

FIG. 4 shows a BMP-3B/GDF-10 precursor amino acid sequence (SEQ ID NO:4). The signal peptide (residues 1-33) is underlined; the prodomain(residues 34-368) is in bold, also referred to as SEQ ID NO: 2; and themature protein (residues 369-478) is shaded. The potential N-linkedglycosylation sites are boxed.

FIG. 5 shows a nucleic acid sequence encoding a BMP-3A propeptide,designed as SEQ ID NO: 5.

FIG. 6 shows a nucleic acid sequence encoding a BMP-3B propeptide,designed as SEQ ID NO:6.

FIG. 7 shows a nucleic acid sequence encoding the BMP-3A precursorprotein, designed as SEQ ID NO: 7.

FIG. 8 shows a nucleic acid sequence encoding the BMP-3B precursorprotein, designed as SEQ ID NO: 8.

FIG. 9 shows a human Fc amino acid sequence. Certain optional mutationsare shown in red.

FIG. 10 shows BMP3A propeptide-Murine IgG2a peptide Fc fusion protein(“BMP3 pro-MuIg2a”) purified using Protein A affinity step. Lane 1indicates molecular weight markers, while Lane 2 shows the BMP3-Pro-MuFcfusion purified from conditioned media of cells expressing BMP3pro-MuIgG2a by Protein A affinity purification.

FIG. 11 shows that BMP3 pro-MuIgG2a peptide binds mature BMP-3. Lane 1:Molecular Weight Markers. Lane 2: Control-incubation of protein A beadswith conditioned media from 293 cells transfected with vector alone andmature BMP-3. Lane 3: Conditioned media from HEK293 cell expressing BMP3pro-MuIgG2α-peptide incubated with BMP-3 and Protein A.

FIG. 12 shows binding of purified BMP3 pro-MuIgG2a to mature BMP-3 usingBiaCore CM5 chip analysis. Purified BMP3 pro-MuIgG2a was coupled onto aBiaCore CM5 chip using the amine coupling procedure. Panel A: MatureBMP-3 was flowed over the chip. Panel B: Control experiment showing thatBSA does not bind to BMP3 pro-MuIgG2a Fc.

FIG. 13 shows that mature BMP-3 binds to Activin Receptor Ia (ActRIIa)and BMP3 pro-MuIgG2a Fc competes with ActRIIa binding to BMP-3. ActRIIawas immobilized on a BiaCore CM5 chip using standard amine couplingprocedure. Trace A: Mature BMP-3 (200 μg/ml) was injected on the ActRIIacoupled chip. Trace B: BMP3 pro-MuIgG2a Fc (100 μg/ml) was injected ontothe ActRIIa chip (no binding). Trace C: Mature BMP-3 (200 μg/ml) andBMP3 pro-MuIgG2a (100 μg/ml)) were premixed and injected onto theActRIIa coupled chip.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention relates to Bone morphogenetic protein-3 (BMP-3)propeptides. As used herein, the term “BMP-3” refers to the family ofbone morphogenetic proteins of the type 3, derived from any species.Reference to BMP-3 herein is understood to be a reference to any one ofthe currently identified forms, including BMP-3A (also known as BMP-3)and BMP-3B (also known as GDF-10), as appropriate from the context. Theterm “BMP-3” also includes polypeptides derived from the sequence of anyknown BMP-3 whose mature sequence is at least about 75% homologous withthe sequence of a mature BMP-3, and preferably at least 80%, 85%, 90%,95%, 97%, 99% or greater homology. Members of the BMP-3 family aregenerally encoded as a larger precursor, and members of the family sharea region of high homology near the C-terminus, corresponding generallyto the mature portion. For example, BMP-3B shares 82% amino acididentity with BMP-3A in the mature peptide (mature domain), but only 37%in the propeptide. Note that because the mature proteins sharesubstantial homology, it is expected that the BMP-3A propeptide willbind to either mature protein.

A naturally occurring BMP-3 protein is generally encoded as a largerprecursor that typically contains a signal sequence at its N-terminusfollowed by a dibasic amino acid cleavage site and a propeptide,followed by another dibasic amino acid cleavage site and a maturedomain. Thus, as used herein, the term “propeptide” or “prodomain” isthe portion that is N-terminal to the mature domain and C-terminal tothe signal peptide. Optionally, a BMP-3 propeptide, after cleavage,reassociates with its mature peptide covalently or non-covalently, as inthe case of insulin, relaxin, inhibin, activin, and TGF-β. The term“BMP-3 propeptide” is used to refer to polypeptides comprising anynaturally occurring propeptide of a BMP-3 family member as well as anyvariants thereof (including mutants, fragments and peptidomimetic forms)that retain a useful activity. As used herein, BMP-3 propeptides includefragments, functional variants, and modified forms (e.g., peptidomimeticforms) of BMP-3 propeptides. A “BMP-3 propeptide” will not include afull-length mature BMP-3 domain, although a BMP-3 propeptide may includeportions of the mature domain, particularly portions that are not fullyfunctional. For example, a BMP-3 propeptide may contain fewer than 50,40, 30, 20, 10 or 5 amino acids of its cognate mature domain. Functionalvariants of a BMP-3 propeptide may be characterized by, for example,binding to mature BMP-3 protein and/or the ability to competitivelyinhibit the binding of BMP-3 to a type II receptor, such as ActRIIa.

BMP-3A and BMP-3B together represent a unique subgroup of the BMPfamily. In osteoblasts, BMP-3A and 3B have been shown to antagonize theBMP-2 activity (Hino et al., 2004, Front Biosci., 9:1520-9; Daluiski etal, 2001, Nature Genetics, 27:84-88). BMP-2 is known to promote boneformation and mineralization; BMP-2 is reported to stimulate osteoblastsand is also reported to promote the production of osteoblasts fromprogenitor cells. While not wishing to be bound to any particularmechanism, it is expected that a BMP-3 propeptide promotes bone growth(meaning increased bone density, volume or other appropriate metric) byantagonizing the mature BMP-3, thus dis-inhibiting one or more BMP-2functions. BMP-3 may also have independent effects, perhaps stimulatingosteoclasts, and thus causing increased bone resorbtion. The sequencesof BMP-3A and BMP-3B precursor proteins (i.e., signal peptide,propeptide, and mature peptide) are illustrated in FIG. 3 and FIG. 4,respectively.

As described in the working examples, Applicants have demonstrated thata BMP-3 propeptide (e.g., a BMP-3 Fc fusion protein) binds to matureBMP-3A and competes with mature BMP-3A for binding to the ActivinReceptor IIa (ActRIIa). Accordingly, in certain embodiments,compositions and methods of the present invention are useful forinhibiting interaction between mature BMP-3A and ActRIIa. In furtherembodiments, compositions and methods of the present invention areuseful for inhibiting any signaling mediated by interaction betweenmature BMP-3A and ActRIIa.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The terms “bone loss” and “bone growth” are used herein to refer tochanges (decreases or increases, respectively) in size or density ofbone measured in any way, such as changes in bone volume, density ormineralization. For example, these characteristics may be assessed interms of rates of loss or growth or in terms of snapshot (single timepoint) or equilibrium comparisons.

The disclosure may refer to the comparison of sequences to each other,including the comparison of wild-type sequence to one or moremutants/sequence variants. Such comparisons typically comprisealignments of polymer sequences, e.g., using sequence alignment programsand/or algorithms that are well known in the art (for example, BLAST,FASTA and MEGALIGN, to name a few). The skilled artisan can readilyappreciate that, in such alignments, where a mutation contains a residueinsertion or deletion, the sequence alignment will introduce a “gap”(typically represented by a dash, or “A”) in the polymer sequence notcontaining the inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. BMP-3 Propeptides

In certain aspects, the invention relates to BMP-3 propeptides.Preferably, these fragments, functional variants, and modified formshave biological activities that are similar to or the same as that oftheir corresponding wild-type BMP-3 propeptides. For example, a BMP-3propeptide of the invention binds to and inhibits a function of a matureBMP-3 protein. In certain cases, the BMP-3 propeptide inhibits theinteraction between a mature BMP-3 polypeptide and an ActRIIapolypeptide. In other cases, the BMP-3 propeptide inhibits any signalingmediated by interaction between a mature BMP-3 polypeptide and anActRIIa polypeptide. Optionally, a BMP-3 propeptide derepresses orincreases bone growth.

Examples of BMP-3 propeptides include a BMP-3A propeptide (SEQ ID NO: 1)and a BMP-3B propeptide (SEQ ID NO: 2). Although the BMP-3A and 3Bpropeptides are only distantly similar in amino acid sequence, eachbinds to a mature polypeptide of similar sequence, and accordingly it isexpected that BMP-3B propeptide binds BMP-3A and vice versa.

In one specific example, human BMP-3A cDNA (SEQ ID NO: 7, FIG. 7)encodes a 472-amino acid precursor protein (SEQ ID NO: 3, FIG. 3).Cleavage of the BMP-3A precursor protein at a putative polybasicproteolytic cleavage site (residues 357-362 of SEQ ID NO: 3) generates amature BMP-3A protein consisting of 110 amino acids (FIG. 3) and aBMP-3A propeptide consisting of 340 amino acids (FIGS. 1 and 3, SEQ IDNO: 1). The BMP-3A propeptide contains potential glycosylation sites(FIG. 3). See, e.g., Wozney et al., 1988, Science. 242:1528-34.

In another specific example, human BMP-3B cDNA (SEQ ID NO: 8, FIG. 8)encodes a 478-amino acid precursor protein. Cleavage of the BMP-3Bprecursor protein at a putative polybasic proteolytic cleavage site(residues 364-368 of SEQ ID NO: 4) generates a mature BMP-3B proteinconsisting of 110 amino acids and a BMP-3B propeptide consisting of 335amino acids (FIGS. 2 and 4, SEQ ID NO: 2). Both the BMP-3B propeptideand the BMP-3B mature peptide contain potential glycosylation sites(FIG. 4). See, e.g., Hino et al., 1996, Biochem. Biophys. Res. Commun.223 (2), 304-310.

In certain embodiments, isolated fragments of the BMP-3 propeptides canbe obtained by screening polypeptides recombinantly produced from thecorresponding fragment of the nucleic acid encoding a BMP-3 propeptide(e.g., SEQ ID NO: 1 or 2). In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. The fragments can beproduced (recombinantly or by chemical synthesis) and tested to identifythose peptidyl fragments that can function to stimulate bone growth, forexample, as antagonists of the BMP-3 activity or as activator of BMP-2activity.

In certain embodiments, a functional variant of the BMP-3 propeptideshas an amino acid sequence that is at least 75% identical to an aminoacid sequence as set forth in SEQ ID NO: 1 or 2. In certain cases, thefunctional variant has an amino acid sequence at least 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to an amino acid sequence as setforth in SEQ ID NO: 1 or 2. Preferably such variants retain the abilityto bind to mature BMP-3 and/or competitively inhibit the binding ofBMP-3 to a type II receptor.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of a BMP-3 propeptide.Such modifications may be made, for example, for such purposes asenhancing therapeutic efficacy, or stability (e.g., ex vivo shelf lifeand resistance to proteolytic degradation in vivo). Such modified BMP-3propeptides when designed to retain at least one activity of thenaturally-occurring form of the BMP-3 propeptides, are consideredfunctional equivalents of the naturally-occurring propeptides. ModifiedBMP-3 propeptides can also be produced, for instance, by amino acidsubstitution, deletion, or addition. For instance, it is reasonable toexpect that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of a BMP-3 propeptide results in a functional homolog can bereadily determined by assessing the ability of the variant propeptide toproduce a response in cells in a fashion similar to the wild-typepropeptide.

In certain embodiments, the present invention contemplates makingmutations in the proteolytic cleavage site of the BMP-3 propeptidesequence to make the site less susceptible to proteolytic cleavage.Computer analysis (using a commercially available software, e.g.,MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used toidentify proteolytic cleavage sites. As will be recognized by one ofskill in the art, most of the described mutations, variants ormodifications may be made at the nucleic acid level or, in some cases,by post translational modification or chemical synthesis. Suchtechniques are well known in the art.

In certain embodiments, the present invention contemplates specificmutations of the BMP-3 propeptide sequences so as to alter theglycosylation of the polypeptide. Such mutations may be selected so asto introduce or eliminate one or more glycosylation sites, such asO-linked or N-linked glycosylation sites. Asparagine-linkedglycosylation recognition sites generally comprise a tripeptidesequence, asparagine-X-threonine (where “X” is any amino acid) which arespecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type BMP-3 propeptide (for O-linked glycosylation sites). A varietyof amino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Another means of increasing thenumber of carbohydrate moieties on a BMP-3 propeptide is by chemical orenzymatic coupling of glycosides to the BMP-3 propeptide. Depending onthe coupling mode used, the sugar(s) may be attached to (a) arginine andhistidine; (b) free carboxyl groups; (c) free sulfhydryl groups such asthose of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein. Removal of one or morecarbohydrate moieties present on a BMP-3 propeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the BMP-3 propeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on BMP-3 propeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al. (1987) Meth.Enzymol. 138:350. The nucleic acid and/or amino acid sequence of apropeptide may be adjusted, as appropriate, depending on the type ofexpression system used, as mammalian, yeast, insect and plant cells mayall introduce differing glycosylation patterns that can be affected bythe amino acid sequence of the peptide.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of the BMP-3 propeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, BMP-3 propeptide variants which can act as either agonistsor antagonist, or alternatively, which possess novel activities alltogether. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, a BMP-3 propeptidevariant may be screened for ability to bind to a mature BMP-3polypeptide, or for the ability to prevent binding of a mature BMP-3 toa cell expressing a BMP-3 receptor, such as an ActRII. The activity of aBMP-3 may also be tested in a cell-based or in vivo assay. For example,the effect of a BMP-3 propeptide on the expression of genes involved inbone production in an osteoblast or precursor may be assessed. This may,as needed, be performed in the presence of recombinant BMP-3 and/orBMP-2, and cells may be transfected so as to produce any of BMP-2, BMP-3and the subject BMP-3 propeptide variant. Likewise, a BMP-3 propeptidemay be administered to a mouse or other animal, and one or more boneproperties, such as density or volume may be assessed. The healing ratefor bone fractures may also be evaluated.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring BMP-3 propeptide. Such variantproteins, when expressed from recombinant DNA constructs, can be used ingene therapy protocols. Likewise, mutagenesis can give rise to variantswhich have intracellular half-lives dramatically different than thecorresponding wild-type propeptide. For example, the altered protein canbe rendered either more stable or less stable to proteolytic degradationor other cellular process which result in destruction of, or otherwiseinactivation of a native BMP-3 propeptide. Such variants, and the geneswhich encode them, can be utilized to alter BMP-3 propeptide levels bymodulating the half-life of the propeptide. For instance, a shorthalf-life can give rise to more transient biological effects and, whenpart of an inducible expression system or scheduled dosing regimen, canallow tighter control of recombinant BMP-3 propeptide levels in thetreated subject.

In a preferred embodiment, the combinatorial library is produced by wayof a degenerate library of genes encoding a library of polypeptideswhich each include at least a portion of potential BMP-3 propeptidesequences. For instance, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential BMP-3 propeptide nucleotide sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then be ligated into an appropriatevector for expression. The synthesis of degenerate oligonucleotides iswell known in the art (see for example, Narang, S A (1983) Tetrahedron39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd ClevelandSympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp 273-289;Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,(1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, BMP-3 propeptide variants (bothagonist and antagonist forms) can be generated and isolated from alibrary by screening using, for example, alanine scanning mutagenesisand the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al.,(1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601;Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.,(1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989)Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al.,(1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell. Biol.12:2644-2652; McKnight et al., (1982) Science 232:316); by saturationmutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis(Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by randommutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992)A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linkerscanning mutagenesis, particularly in a combinatorial setting, is anattractive method for identifying truncated (bioactive) forms of BMP-3propeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of BMP-3 propeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, the BMP-3 propeptides of the present inventioninclude peptidomimetics. As used herein, the term “peptidomimetic”includes chemically modified peptides and peptide-like molecules thatcontain non-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the BMP-3 propeptides.

To illustrate, by employing scanning mutagenesis to map the amino acidresidues of a BMP-3 propeptide which are involved in binding to anotherprotein, peptidomimetic compounds can be generated which mimic thoseresidues involved in binding. For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al., in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine(e.g., see Huffman et al., in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgamma lactam rings (Garvey et al., in Peptides: Chemistry and Biology,G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),keto-methylene pseudopeptides (Ewenson et al., (1986) J. Med. Chem.29:295; and Ewenson et al., in Peptides: Structure and Function(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co.Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al., (1985)Tetrahedron Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans1:1231), and b-aminoalcohols (Gordon et al., (1985) Biochem Biophys ResCommun 126:419; and Dann et al., (1986) Biochem Biophys Res Commun134:71).

In certain embodiments, the BMP-3 propeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the propeptide. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. As a result,the modified BMP-3 propeptides may contain non-amino acid elements, suchas polyethylene glycols, lipids, poly- or mono-saccharide, andphosphates. Effects of such non-amino acid elements on the functionalityof a BMP-3 propeptide may be tested as described herein for other BMP-3propeptide variants. When a BMP-3 propeptide is produced in cells bycleaving a nascent form of the BMP-3 protein, post-translationalprocessing may also be important for correct folding and/or function ofthe protein. Different cells (such as CHO, HeLa, MDCK, 293, WI38,NIH-3T3 or HEK293) have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the BMP-3 protein intoa BMP-3 propeptide.

In certain aspects, functional variants or modified forms of the BMP-3propeptides include fusion proteins having at least a portion of theBMP-3 propeptides and one or more fusion domains. Well known examples ofsuch fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the BMP3 propeptide. Examplesof such detection domains include the various fluorescent proteins(e.g., GFP) as well as “epitope tags,” which are usually short peptidesequences for which a specific antibody is available. Well known epitopetags for which specific monoclonal antibodies are readily availableinclude FLAG, influenza virus hemagglutinin (HA), and c-myc tags. Insome cases, the fusion domains have a protease cleavage site, such asfor Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, a BMP-3 propeptide isfused with a domain that stabilizes the propeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.In addition, Fc fusions tend to dimerize, providing a dimeric BMP-3propeptide. Likewise, fusions to human serum albumin can conferdesirable properties. Other types of fusion domains that may be selectedinclude multimerizing (e.g., dimerizing, tetramerizing) domains andfunctional domains (that confer an additional biological function, suchas further stimulation of bone growth).

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a BMP-3 propeptide may be placed C-terminalto a heterologous domain, or, alternatively, a heterologous domain maybe placed C-terminal to a BMP-3 propeptide. The propeptide domain andthe heterologous domain need not be adjacent in a fusion protein, andadditional domains or amino acid sequences may be included C- orN-terminal to either domain or between the domains.

In certain embodiments, the BMP-3 propeptides of the present inventioncontain one or more modifications that are capable of stabilizing theBMP-3 propeptides. For example, such modifications enhance the in vitrohalf life of the propeptides, enhance circulatory half life of thepropeptides or reducing proteolytic degradation of the propeptides. Suchstabilizing modifications include, but are not limited to, fusionproteins (including, for example, fusion proteins comprising a BMP-3propeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to aBMP-3 propeptide), and modifications of carbohydrate moiety (including,for example, removal of carbohydrate moieties from a BMP-3 propeptide).In the case of fusion proteins, a BMP-3 propeptide is fused to astabilizer domain such as an IgG molecule (e.g., an Fc domain). As usedherein, the term “stabilizer domain” not only refers to a fusion domain(e.g., Fc) as in the case of fusion proteins, but also includesnonproteinaceous modifications such as a carbohydrate moiety, ornonproteinaceous polymer, such as polyethylene glycol (PEG). PEG may beaffixed to BMP-3 propeptides in a variety of sizes, ranging from 1000 Dto 50,000 D or more molecular weight polymers. PEG polymers may beaffixed to propeptides in a selective, residue specific manner,particularly when directed against the N-terminal amine or an engineeredcysteine. PEG polymers may also be affixed in a relatively uncontrolledreaction, in which primary amines and/or sulfhydryl groups may bereacted. The stoichiometry may range from 1:1 (PEG:Propeptide) to 2:1and higher.

In certain embodiments, the BMP-3 propeptide is fused with animmunoglobulin Fc domain. In a preferred embodiment, the Fc domain is anIgG1 Fc fragment. An IgG1 Fc fragment may include various alterations,including, for example, mutations that reduce binding to Fcγ Receptorand mutations that decreased binding to MHC class I-related Fc-receptor(FcRN). Examples of mutations include mutations in the an Fc portion atpositions 265 (Asp to Ala), 322 (Lys to Ala), and 434 (Asn to Ala). Anexample of an Fc sequence is shown in FIG. 9.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the BMP-3 propeptides, which are isolated from,or otherwise substantially free of, other proteins.

In certain embodiments, BMP-3 propeptides (unmodified or modified) ofthe invention can be produced by a variety of art-recognized techniques.For example, such BMP-3 propeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the BMP-3 propeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified BMP-3 propeptides maybe produced by digestion of naturally occurring or recombinantlyproduced BMP-3 by using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using a commercially availablesoftware, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.)can be used to identify proteolytic cleavage sites. Alternatively, suchpropeptides may be produced from naturally occurring or recombinantlyproduced BMP-3 such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

In certain embodiments, the present invention contemplates makingmutations in the proteolytic cleavage site of the BMP-3 sequence to makethe site less susceptible to proteolytic cleavage. The result is a BMP-3polypeptide containing both propeptide and mature portion, which may beuseful as a BMP-3 antagonist. More preferably, the mature portion isengineered with a stop codon, such that the BMP-3 propeptide is producedwith some portion of the mature peptide attached. In one specificembodiment, a mutant may contain a point mutation at amino acids 357,358, 359, 360, 361 or 362 of SEQ ID NO: 3, or at amino acids 335, 336,337, 338, 339 or 340 of SEQ ID NO: 1. In another specific embodiment,such mutant may contain a point mutation at amino acids 364, 365, 366,367 or 368 of SEQ ID NO: 4, or at amino acids 331, 332, 333, 334 or 335of SEQ ID NO: 2.

3. Nucleic Acids Encoding BMP-3 Propeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the BMP-3 propeptides, includingfunctional variants, disclosed herein. For example, SEQ ID NOs: 5 and 6encode BMP-3 propeptides. The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids are may be used, for example, in methodsfor making BMP-3 propeptides or as direct therapeutic agents (e.g., in agene therapy approach).

The subject nucleic acids encoding BMP-3 propeptides are furtherunderstood to include nucleic acids that are variants of SEQ ID NOs: 5and 6. Variant nucleotide sequences include sequences that differ by oneor more nucleotide substitutions, additions or deletions, such asallelic variants; and will, therefore, include coding sequences thatdiffer from the nucleotide sequence of the coding sequence designated inSEQ ID NOs: 5 and 6.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 5 or 6. One of ordinary skill in theart will appreciate that nucleic acid sequences complementary to SEQ IDNO: 5 or 6, and variants of SEQ ID NO: 5 or 6 are also within the scopeof this invention. In further embodiments, the nucleic acid sequences ofthe invention can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 5 or 6, complementsequence of SEQ ID NO: 5 or 6, or fragments thereof. As discussed above,one of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 5-6 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a BMP-3 propeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the BMP-3 propeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding a BMP-3 propeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant BMP-3 propeptides include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and transformation of hostorganisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant SLC5A8 polypeptide by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the B-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production of asubject BMP-3 propeptide in CHO cells, such as a Pcmv-Script vector(Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad,Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will beapparent, the subject gene constructs can be used to cause expression ofthe subject BMP-3 propeptide in cells propagated in culture, e.g., toproduce proteins, including fusion proteins or variant proteins, forpurification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 5 or 6)for one or more of the subject BMP-3 propeptides. The host cell may beany prokaryotic or eukaryotic cell. For example, a BMP-3 propeptide ofthe invention may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present invention further pertains to methods ofproducing the subject BMP-3 propeptides. For example, a host celltransfected with an expression vector encoding a BMP-3 propeptide can becultured under appropriate conditions to allow expression of the BMP-3propeptide to occur. The BMP-3 propeptide may be secreted and isolatedfrom a mixture of cells and medium containing the propeptide.Alternatively, the propeptide may be retained cytoplasmically or in amembrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The propeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins, includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for particular epitopes of the propeptide. In apreferred embodiment, the BMP-3 propeptide is a fusion proteincontaining a domain which facilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant BMP-3propeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified BMP-3 propeptide (e.g., see Hochuliet al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Antibodies

Another aspect of the invention pertains to antibodies. An antibody thatis specifically reactive with a mature BMP-3 polypeptide and which bindscompetitively with the BMP-3 propeptide may be used as a BMP-3antagonist. For example, by using immunogens derived from a BMP-3 maturepeptide, anti-protein/anti-peptide antisera or monoclonal antibodies canbe made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the BMP-3 peptide, an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusion protein.In a preferred embodiment, the inoculated mouse does not expressendogenous BMP3, thus facilitating the isolation of antibodies thatwould otherwise be eliminated as anti-self antibodies. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of a BMP-3 peptide can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayscan be used with the immunogen as antigen to assess the levels ofantibodies.

Following immunization of an animal with an antigenic preparation of aBMP-3, antisera can be obtained and, if desired, polyclonal antibodiescan be isolated from the serum. To produce monoclonal antibodies,antibody-producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique (originally developed by Kohler and Milstein, (1975)Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar etal., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with BMP-3 and monoclonal antibodies isolated froma culture comprising such hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject BMP-3peptide. Antibodies can be fragmented using conventional techniques andthe fragments screened for utility in the same manner as described abovefor whole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments. Theantibody of the present invention is further intended to includebispecific, single-chain, and chimeric and humanized molecules havingaffinity for a BMP-3 peptide conferred by at least one CDR region of theantibody. In preferred embodiments, the antibody further comprises alabel attached thereto and able to be detected (e.g., the label can be aradioisotope, fluorescent compound, enzyme or enzyme co-factor).

In certain preferred embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments, the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to a BMP-3peptide may comprise administering to a mouse an amount of animmunogenic composition comprising the BMP-3 propeptide effective tostimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monocolonal antibody that binds specificallyto the BMP-3 peptide. Once obtained, a hybridoma can be propagated in acell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the BMP-3 peptide. The monoclonal antibody may bepurified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., a BMP-3 peptide) and other antigens that are not ofinterest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure provides antibodies that bind to aBMP-3 propeptide. Such antibodies may be generated much as describedabove, using a propeptide or fragment thereof as an antigen. Antibodiesof this type can be used, e.g., to detect BMP-3 propeptides inbiological samples and/or to monitor BMP-3 propeptide levels in anindividual. The level of BMP-3 propeptides maybe measured in a varietyof sample types such as, for example, in cells, and/or in bodily fluid,such as in whole blood samples, blood serum, blood plasma and urine. Anantibody that binds to a BMP-3 propeptide can be used to stimulate BMP-3activity, encouraging decreased bone growth or bone resorption. This maybe desirable in various disorders, such as any hyperostosis,osteopoikilosis, osteopetrosis, osteosclerosis (e.g., resulting fromrenal insufficiency), pyknodysostosis, osteomyelosclerosis, andhyperphosphatasia.

5. Screening Assays

In certain aspects, the present invention relates to the use of thesubject BMP-3 propeptides to identify compounds (agents) which areagonist or antagonists of the BMP-3 propeptides. Compounds identifiedthrough this screening can be tested in bone and/or cartilage tissues toassess their ability to modulate bone/cartilage growth in vitro.Optionally, these compounds can further be tested in animal models toassess their ability to modulate bone/cartilage growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating bone growth by targeting the BMP-3 propeptides. In certainembodiments, high-throughput screening of compounds can be carried outto identify agents that perturb BMP-3A propeptide-mediated effects onbone or cartilage growth, such by assessing the effects of suchcompounds on BMP-3 propeptide-mediated effects on the osteogenicactivity of BMP-2. In certain embodiments, the assay is carried out toscreen and identify compounds that specifically inhibit or reducebinding of a BMP-3 propeptide to its binding partner (e.g., a BMP-3mature peptide). Alternatively, the assay can be used to identifycompounds that enhance binding of a BMP-3 propeptide to its bindingprotein (e.g., a BMP-3 mature peptide). In a further embodiment, thecompounds can be identified by their ability to interact with a BMP-3propeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof bone or cartilage growth can be produced, for example, by bacteria,yeast, plants or other organisms (e.g., natural products), producedchemically (e.g., small molecules, including peptidomimetics), orproduced recombinantly. Test compounds contemplated by the presentinvention include non-peptidyl organic molecules, peptides,polypeptides, peptidomimetics, sugars, hormones, and nucleic acidmolecules. In a specific embodiment, the test agent is a small organicmolecule having a molecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase,photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a BMP-3propeptide and its binding protein (e.g., a BMP-3 mature peptide).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified BMP-3 propeptide which is ordinarily capable of binding to aBMP-3 mature peptide, as appropriate for the purpose of the assay. Tothe mixture of the compound and BMP-3 propeptide is then added acomposition containing a BMP-3 mature peptide. Detection andquantification of BMP-3 propeptide complexes provides a means fordetermining the compound's efficacy at inhibiting (or potentiating)complex formation between the BMP-3 propeptide and its binding protein(e.g., a BMP-3 mature peptide). The efficacy of the compound can beassessed by generating dose response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. For example,in a control assay, isolated and purified BMP-3 mature peptide is addedto a composition containing the BMP-3 propeptide, and the formation ofBMP-3 propeptide/mature peptide complex is quantitated in the absence ofthe test compound. It will be understood that, in general, the order inwhich the reactants may be admixed can be varied, and can be admixedsimultaneously. Moreover, in place of purified proteins, cellularextracts and lysates may be used to render a suitable cell-free assaysystem.

Complex formation between the BMP-3 propeptide and its binding proteinmay be detected by a variety of techniques. For instance, modulation ofthe formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabelled (e.g., ³²P, ³⁵S, ¹⁴Cor ³H), fluorescently labeled (e.g., FITC), or enzymatically labeledBMP-3 propeptide or its binding protein, by immunoassay, or bychromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a BMP-3 propeptide and its binding protein(e.g., a BMP-3 mature peptide). Further, other modes of detection suchas those based on optical waveguides (PCT Publication WO 96/26432 andU.S. Pat. No. 5,677,196), surface plasmon resonance (SPR) (the modeemployed by BiaCore systems used in the Examples, below), surface chargesensors, and surface force sensors are compatible with many embodimentsof the invention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a BMP-3 propeptide andits binding protein (e.g., a BMP-3 mature peptide). See for example,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In aspecific embodiment, the present invention contemplates the use ofreverse two hybrid systems to identify compounds (e.g., small moleculesor peptides) that dissociate interactions between a BMP-3 propeptide andits binding protein (e.g., a BMP-3 mature peptide). See for example,Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal andLegrain, (1999) Trends Biotechnol 17:374-81; and U.S. Pat. Nos.5,525,490; 5,955,280; 5,965,368.

In one specific example, interaction between a BMP-3 propeptide and amature BMP-3 peptide can be assayed by making a construct whichexpresses a FLAG-tagged BMP-3 precursor protein. The FLAG-taggedprecursor protein is expressed in cells and processed into a BMP-3propeptide and a FLAG-tagged mature BMP-3 peptide. The protein lysatesprepared from the cells are then affinity-purified by antibodies againstFLAG. Complexes containing a BMP-3 propeptide and a mature BMP-3 peptidecan be determined by the presence of a BMP-3 propeptide in theseaffinity-purified protein samples (e.g., by immunoblot).

In certain embodiments, the subject compounds are identified by theirability to interact with a BMP-3 propeptide of the invention. Theinteraction between the compound and the BMP-3 propeptide may becovalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to a BMP-3propeptide. This may include a solid phase or fluid phase binding event.Alternatively, the gene encoding a BMP-3 propeptide can be transfectedwith a reporter system (e.g., β-galactosidase, luciferase, or greenfluorescent protein) into a cell and screened against the librarypreferably by a high throughput screening or with individual members ofthe library. Other mechanism based binding assays may be used, forexample, binding assays which detect changes in free energy. Bindingassays can be performed with the target fixed to a well, bead or chip orcaptured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor stimulating bone formation and increasing bone mass. Therefore, anycompound identified using a cell-free system, or any other compound thatis expected to affect BMP-3 function (e.g., antagonizing the osteogenicactivity of BMP-2), can be tested in whole cells or tissues, in vitro orin vivo, to confirm their ability to modulate bone or cartilage growth.Various methods known in the art can be utilized for this purpose.

For example, BMP-3A inhibits BMP2-mediated induction of Msx2 and blocksBMP2-mediated differentiation of osteoprogenitor cells into osteoblasts.Thus, the effect of the BMP-3 propeptides or the test compounds on boneor cartilage growth can be determined by their effect on the osteogenicactivity of BMP-2, for example, by measuring induction of Msx2 ordifferentiation of osteoprogenitor cells into osteoblasts in cell basedassays (see, e.g., Daluiski et al., Nat. Genet. 2001, 27(1):84-8; Hinoet al., Front Biosci. 2004, 9:1520-9).

Another example of cell-based assays includes analyzing the osteogenicactivity of the subject BMP-3 propeptides and test compounds inmesenchymal progenitor and osteoblastic cells. To illustrate,recombinant adenoviruses expressing a human BMP-3 propeptide wereconstructed to infect pluripotent mesenchymal progenitor C3H10T1/2cells, preosteoblastic C2C12 cells, and osteoblastic TE-85 cells.Osteogenic activity is then determined by measuring the induction ofalkaline phosphatase, osteocalcin, and matrix mineralization (see, e.g.,Cheng et al., J bone Joint Surg Am. 2003, 85-A(8): 1544-52).

Further, the present invention contemplates in vivo assays to measurebone or cartilage growth. For example, Namkung-Matthai et al., Bone,28:80-86 (2001) discloses a rat osteoporotic model in which bone repairduring the early period after fracture is studied. Kubo et al., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a ratosteoporotic model in which bone repair during the late period afterfracture is studied. These references are incorporated by referenceherein in their entirety for their disclosure of rat model for study onosteoporotic bone fracture. In certain aspects, the present inventionmakes use of fracture healing assays that are known in the art. Theseassays include fracture technique, histological analysis, andbiomechanical analysis, which are described in, for example, U.S. Pat.No. 6,521,750, which is incorporated by reference in its entirety forits disclosure of experimental protocols for causing as well asmeasuring the extent of fractures, and the repair process.

It is understood that the screening assays of the present inventionapply to not only the subject BMP-3 propeptides and variants of theBMP-3 propeptides, but also any test compounds including agonists andantagonist of the BMP-3 propeptides. Further, these screening assays areuseful for drug target verification and quality control purposes.

6. Methods of Administration

In certain embodiments, compositions (e.g., BMP-3 propeptides andcompositions comprising such propeptides) of the present invention canbe used for inducing bone and/or cartilage formation, preventing boneloss, increasing bone mineralization or preventing the demineralizationof bone. For example, the subject BMP-3 propeptides and compoundsidentified in the present invention have application in treatingosteoporosis and the healing of bone fractures and cartilage defects inhumans and other animals. BMP-3 propeptides may be useful in patientsthat are diagnosed with subclinical low bone density, as a protectivemeasure against the development of osteoporosis.

In a certain embodiment, the present invention provides methods oftreating or preventing an individual suffering from a disease (disorderor condition) that is related to bone/cartilage defects throughadministering to the individual a therapeutically effective amount of aBMP-3 propeptide as described above. These methods are particularlyaimed at therapeutic and prophylactic treatments of animals, and moreparticularly, humans.

In certain embodiment, methods and compositions of the present inventionmay find medical utility in the healing of bone fractures and cartilagedefects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma-induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. Further, methods and compositions of the invention maybe used in the treatment of periodontal disease, and in other toothrepair processes. In certain cases, the subject BMP-3 propeptides mayprovide an environment to attract bone-forming cells, stimulate growthof bone-forming cells or induce differentiation of progenitors ofbone-forming cells. BMP-3 propeptides of the invention may also beuseful in the treatment of osteoporosis. Further, BMP-3 propeptides maybe used in cartilage defect repair and prevention/reversal ofosteoarthritis.

In certain embodiment, methods and compositions of the invention mayalso be used in wound healing and related tissue repair. The types ofwounds include, but are not limited to, burns, incisions and ulcers. Seee.g., PCT Publication No. WO84/01106.

In certain embodiment, the invention provides a therapeutic method andcomposition for repairing fractures and other conditions related tocartilage and/or bone defects or periodontal diseases. The inventionfurther provides therapeutic methods and compositions for wound healingand tissue repair. Such compositions comprise a therapeuticallyeffective amount of at least one of the BMP-3 propeptide of theinvention in admixture with a pharmaceutically acceptable vehicle,carrier or matrix.

In certain specific embodiments, methods and compositions (e.g., BMP-3propeptides) of the invention can be applied to conditions causing boneloss such as osteoporosis, hyperparathyroidism, Cushing's disease,thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubularacidosis, or anorexia nervosa. Many people know that being female,having a low body weight, and leading a sedentary lifestyle are riskfactors for osteoporosis (loss of bone mineral density, leading tofracture risk). However, osteoporosis can also result from the long-termuse of certain medications. Osteoporosis resulting from drugs or anothermedical condition is known as secondary osteoporosis. In a conditionknown as Cushing's disease, the excess amount of cortisol produced bythe body results in osteoporosis and fractures. The most commonmedications associated with secondary osteoporosis are thecorticosteroids, a class of drugs that act like cortisol, a hormoneproduced naturally by the adrenal glands. Although adequate levels ofthyroid hormones (which are produced by the thyroid gland) are neededfor the development of the skeleton, excess thyroid hormone can decreasebone mass over time. Antacids that contain aluminum can lead to boneloss when taken in high doses by people with kidney problems,particularly those undergoing dialysis. Other medications that can causesecondary osteoporosis include phenyloin (Dilantin) and barbituratesthat are used to prevent seizures; methotrexate (Rheumatrex, Immunex,Folex PFS), a drug for some forms of arthritis, cancer, and immunedisorders; cyclosporine (Sandimmune, Neoral), a drug used to treat someautoimmune diseases and to suppress the immune system in organtransplant patients; luteinizing hormone-releasing hormone agonists(Lupron, Zoladex), used to treat prostate cancer and endometriosis;heparin (Calciparine, Liquaemin), an anticlotting medication; andcholestyramine (Questran) and colestipol (Colestid), used to treat highcholesterol. Gum disease causes bone loss in part because of the effectsof bacterial toxins and in part because of the effects of prolongedinflammation.

In certain embodiments, the present invention provides methods andtherapeutic agents, for example, antagonists of BMP-3 propeptides, fortreating diseases or disorders associated with abnormal or unwanted bonegrowth. For example, patients having the disease known as FibrodysplasiaOssificans Progressiva (FOP) grow an abnormal “second skeleton” thatprevents any movement. Additionally, abnormal bone growth can occurafter hip replacement surgery and thus ruin the surgical outcome. Thisis a more common example of pathological bone growth and a situation inwhich antagonists of BMP-3 propeptides may be therapeutically useful.Antagonists of BMP-3 propeptides may also be useful for treating otherforms of abnormal bone growth, such as the pathological growth of bonefollowing trauma, burns or spinal cord injury. In addition, antagonistsof BMP-3 propeptides may be useful for treating or preventing theundesirable actions of BMPs associated with the abnormal bone growthseen in connection with metastatic prostate cancer or osteosarcoma.Examples of these antagonists of BMP-3 propeptides include, but are notlimited to, compounds that disrupt interaction between a BMP-3propeptide and its binding partner (e.g., a BMP-3 mature peptide) andantibodies that specifically bind to a BMP-3 propeptide.

In certain embodiments of the subject methods, one or more BMP-3propeptides can be administered, together (simultaneously) or atdifferent times (sequentially or overlapping). In addition, BMP-3propeptides can be administered with another type of osteogenic,cartilage-inducing or bone-inducing factor. The two types of compoundsmay be administered simultaneously or at different times. It is expectedthat the BMP-3 propeptides of the invention may act in concert with orperhaps synergistically with other osteogenic, cartilage-inducing orbone-inducing factors. A variety of osteogenic, cartilage-inducing andbone-inducing factors have been described, particularly bisphosphonates.See e.g., European Patent Application Nos. 148,155 and 169,016. Forexample, other factors that can be combined with the subject BMP-3propeptides include various growth factors such as epidermal growthfactor (EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., BMP-3 propeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, a BMP-3 propeptide (including, for example, an Fcfusion protein thereof) can be administered alone or as a component of apharmaceutical formulation (therapeutic composition). The subjectcompounds may be formulated for administration in any convenient way foruse in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is generally in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to the site of bone,cartilage or tissue damage. Topical administration may be suitable forwound healing and tissue repair. Therapeutically useful agents otherthan the BMP-3 propeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the BMP-3 propeptidesin the methods of the invention. Preferably for bone and/or cartilageformation, the composition would include a matrix capable of deliveringthe BMP-3 propeptides or other therapeutic compounds to the site of boneand/or cartilage damage, providing a structure for the developing boneand cartilage and optimally capable of being resorbed into the body. Forexample, the matrix may provide slow release of the BMP-3 propeptides.Such matrices may be formed of materials presently in use for otherimplanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., a BMP-3 propeptide), excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more BMP-3 propeptides incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician or veterinarian considering various factors whichmodify the action of the subject compounds of the invention (e.g., BMP-3propeptides). The various factors include, but are not limited to,amount of bone weight desired to be formed, the site of bone damage, thecondition of the damaged bone, the size of a wound, type of damagedtissue, the patient's age, sex, and diet, the severity of any infection,time of administration, and other clinical factors. Optionally, thedosage may vary with the type of matrix used in the reconstitution andthe types of compounds in the composition. The addition of other knowngrowth factors to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of bone growth and/orrepair, for example, X-rays, histomorphometric determinations, andtetracycline labeling.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Construction Expression, and Purification of BMP3 pro-MuIgG2a(BMP3 pro) Fusion

A BMP3 propeptide (BMP3-pro) sequence was PCR amplified from a fulllength BMP3 cDNA and cloned into a human CMV derived expression vectorin such a way that upon ligation it gave a fusion peptide with murineIgG2a. This construct (referred to as BMP3 pro-MuIgG2a) was transientlytransfected in HEK293 cells using polyethylenimine (PEI). After sevendays, cells were harvested and conditioned media was collected forpurification.

Recombinant murine BMP3 pro-MuIgG2a fusion was expressed in HEK293 cellsand purified by protein A affinity chromatography. 1 liter batch ofconditioned media was filtrated, concentrated and loaded on a 4 mlrProtein A Sepharose Fast Flow column (Amersham Biosciences) previouslyequilibrated with TBS (pH 8.0). After protein loading, the column waswashed with 20 column volumes (CV) of TBS, 10 CV of TBS-0.05% Tween 20,followed by additional wash with 10 CV of TBS to remove non-specificallybound proteins. Bound BMP3 pro-MuIgG2a protein was eluted with 100 mMGlycine (pH 3.0). Eluted fraction was immediately neutralized byaddition of 1 M Tris and dialyzed against PBS (pH 8.0). Proteinconcentration was determined by BSA Protein Assay (Pierce). Proteinpurity was determined by 4-12% SDS-PAGE using pre-cast gels (Invitrogen)followed by Sure Blue staining (Invitrogen). The purified BMP3pro-MuIgG2a migrates as a broad band on 4-12% SDS-PAGE gel, whichindicates high degree of glycosylation. Purified protein wasadditionally analyzed by size exclusion chromatography on Superose TMcolumn (Pharmacia Biotech). LAL assay was performed on the purified BMP3pro-MuIgG2a and the amount of endotoxin detected was <0.5 U.

FIG. 10 shows BMP3 pro-MuIgG2a peptide Fc purified using Protein Aaffinity step. Lane 1 indicates molecular weight markers, while Lane 2shows BMP3 pro-MuIgG2a peptide Fc purified from conditioned mediaexpressing BMP3 pro-MuIgG2a peptide Fc using Protein A.

Example 2 BMP3 pro-MuIgG2a Fusion Peptide Binds to Mature BMP-3

Immunoprecipitation studies were first carried out. Conditioned mediawas incubated with protein A beads, washed (3×) with Tris bufferedsaline containing 0.5% Tween 20 (TBST) to remove unbound protein. MatureBMP-3 was added and incubated overnight at 4° C. The beads were washed(3×) with TBST. The beads were resuspended in SDS sample buffercontaining reducing agent and western blot analysis was performed withan anti-mature BMP-3 monoclonal antibody and detected with a HRP labeledanti-mouse IgG. As shown in FIG. 11, BMP3 pro-MuIgG2a peptide binds tomature BMP-3. Lane 1: Molecular Weight Markers. Lane 2:Control-incubation of protein A beads with conditioned media from 293cells transfected with vector alone and mature BMP-3. Lane 3:Conditioned media from HEK293 cell expressing BMP3 pro-MuIgG2a-peptideincubated with BMP-3 and Protein A.

BiaCore chip analysis was also carried out. Purified BMP3 pro-MuIgG2awas coupled onto a BiaCore CM5 chip using the amine coupling procedure.As shown in FIG. 12, purified BMP3 pro-MuIgG2a binds to mature BMP-3using BiaCore CM5 chip analysis. Mature BMP-3 was flowed over the chipand bound to BMP3 pro-MuIgG2a Fc (Panel A). In a control experiment, BSAwas flowed over the chip but did not bind to BMP3 pro-MuIgG2a Fc (PanelB).

Example 3 Mature BMP-3 Binds to Activin Receptor Ia (ActRIIa) and BMP3pro-MuIgG2a Fc Competes with ActRIIa binding to BMP-3

ActRIIa was immobilized on a BiaCore CM5 chip using standard aminecoupling procedure. As shown in FIG. 13, mature BMP-3 (200 μg/ml) wasinjected on the ActRIIa coupled chip. BMP3 pro-MuIgG2a Fc (100 μg/ml)was injected onto the ActRIIa chip. Mature BMP-3 (200 μg/ml) and BMP3pro-MuIgG2a (100 μg/ml)) were premixed and injected onto the ActRIIacoupled chip. As shown in FIG. 13, mature BMP-3 binds to ActRIIa (TraceA); BMP3 pro-MuIgG2a does not bind to ActRIIa (Trace B); and BMP3pro-MuIgG2a Fc competes with ActRIIa binding to BMP-3 (Trace C).

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. An antibody that binds to a mature BMP3 peptide and competes withBMP3 propeptide for binding to the mature BMP3 peptide.
 2. The antibodyof claim 1, wherein the mature BMP3 peptide consists of the sequence ofamino acids 363-472 of SEQ ID NO:3.
 3. The antibody of claim 1, whereinthe antibody comprises at least one CDR region that confers binding tothe mature BMP3 peptide.
 4. The antibody of claim 1, wherein theantibody is a humanized or fully human antibody.
 5. A pharmaceuticalpreparation comprising an antibody of claim 1.